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Advances in Diagnostic Tests for Tuberculosis
Overview of the Diagnostics Pipeline
Richard O’Brien
3rd Global Laboratory Initiative (GLI) Meeting
Veyrier-du-Lac, France
4-5 October 2010
50
195
90
105
105
195
392
392
2006
2007
2008
2009
2010
2011
2012
2013
0 50 100 150 200 250 300 350 400 450
Lives Saved (Thousands/Year)
Ye
ar
of
ST
AG
ap
pro
va
l
Hain
LED FM
MGIT
Capilia
Automated NAAT
Manual NAAT
Ab Assay
Ag Assay
Mol POC450
Potential Impact of New TB Diagnostics
TB Diagnostics Pathway*
*Stop TB New Diagnostics Working Group: A blueprint for the
development of TB Diagnostics, 2009
WHO Policy on Implementing New Tools*
• Identifying the need for a policy change
– WHO monitoring of technical developments
– Requests from interested outside parties
• Reviewing the evidence
– Use of standardized criteria for assessing available data
– Systematic review and meta-analyses
– GRADE approach for rating the strength of a
recommendation
• Convening an expert panel to review evidence and draft
recommendations
• Assessing draft policy and evidence by STAG-TB
• Formulating and disseminating policy
*http://www.who.int/tb/advisory_bodies/research_to_policy/en/index.html
2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
Dis
tan
ce f
rom
Pati
en
ts
Peripheral Lab
First Referral Level
Community Health Care
Abbreviations
DST: Drug Susceptibility Test
NAAT: Nucleic Acid Amplification Test
LTBI: Latent TB Infection
POC: Point of Care
MODS: Microscopic observation drug-
susceptibility
NRA: Nitrate reductase assay
CRI: Colorimetric redox indicator assay
LED: Light-emitting diode
LPA: Line probe assay
Technologies or processes endorsed by STAG/WHO
Technologies for which WHO review is in process
10-40%
70%
95%
% A
ccess a
fter 5
years
•Closed system
• Isothermal
•Rapid
•Multiprimer
•Visible readout
Manual Solution: Eiken
Clinical Performance
Sensitivity in
ss+, LJ+
Sensitivity in
ss-, LJ+
Specificity in
LJ-
Lima 97.7% (75/78) 51.8% (14/27) 99.3% (152/153)
Dhaka 98.4% (61/62) 50% (2/4) 97.8% (181/185)
Mbeya 100% (37/37) 41.7% (5/12) 100% (167/167)
Total 97.7% (173/177) 48.8% (21/43) 99.0% (500/505)
“The assay was robust, with high end-point stability and low rates of test failure.
Technicians with no prior molecular experience easily performed the assay
after 1 week of training, and opportunities for further simplification of the assay
were identified”.
*Boehme CC et al. J Clin Microbio 2007;45:1936-40.
LAMP Feasibility Studies V.1.0
Peru, Tanzania & Bangladesh*
sputum
Heat at 90°C
for 5min
LAMP reaction
at 67℃ 40min
Collect 40ul using
transfer device
Detect fluorescence signal
Close caps and let
it stand upside
down for 2 min
Shake
Mix
25ul
35ul
Heating
container
Dropper capAbsorbent tube
Reaction tube
Add 25 – 35 µl
(between lines)
Dried reagent
Rifampicin
Ciprofloxacin
Liquefaction &
decontamination in
transport medium at
room temperature
MDR
SUSCEPTIBLE
Direct application of 2 drops to selective
thin layer agar for incubation in room air
for MDRTB testing & XDRTB screening
Color growth detection & microscopy
confirmation of morphology
MDR-XDRTB Color Test for Regional Laboratories*
Detection Isoniazid
Biosafety similar to sputum microscopy because sputum is smeared
directly onto the plate which is then permanently double-sealed until autoclaving
1 2 3
*Carlton Evans, Welcome Trust, Peru
MDR-XDRTB Color Test Performance (n=214)
Gold standard=culture positive in any test (n=84/214)
Color test had 2% contamination (all fungal)
Median time to positive result was 16 days
COLOUR | Direct MODS
TEST | MDR not-MDR | Total
------------+----------------------+----------
MDR | 9 3 | 12
not-MDR | 1 68 | 69
------------+----------------------+----------
Total | 10 71 | 81
COLOUR | Indirect TEMA
TEST | MDR not-MDR | Total
------------+----------------------+----------
MDR | 8 4 | 12
not-MDR | 1 51 | 52
------------+----------------------+----------
Total | 9 55 | 64
51% 74% 89% 94%
0%
25%
50%
75%
100%
sen
sit
ivit
y v
s. an
y p
osit
ive (
+95%
CI)
ns P=0.3
P<0.01
P<0.001
MDR-XDRTB
COLOUR
TEST
Centrifuge
decontamination
& low-volume
MODS
ZN
microscopy
Centrifuge
decontamination
& thin layer agar
Culture (TLA)
Concurrent Drug Susceptibility Testing
TB diagnostic sensitivity
New Point-of-Care Technologies
• Antigen and antibody detection
• VOC detection systems
Lipoarabinomannan (LAM)-A (not so) low hanging fruit
• Major cell wall glycolipid of M. tuberculosis
• Described by several groups as diagnostic for TB
• Measurement in urine (+ sputum)
• FIND evaluation of ChemBio / Inverness ELISA
• FIND‘s collaboration with various partners to prove LAM as diagnostic marker
Point of care TB antigen detection: Lipoarabinomannan (LAM) as candidate marker
Sensitivityin ss+ TB
Sensitivity in ss- TB Specificity Non-TB
Specificity healthy
62%
(75/119)
28%
(22/80)
93%
(240/258)
99%
(222/224)
HIV+ HIV - HIV+ HIV -
79% 42% 32% 25%
Chemogen / Inverness assay suggested for urine testing
Claims not supported by study data (4+ independent studies)
TbHC Hospital
staff
Inverness Medical Innovations Launches Clearview TB
ELISA at IAS Meeting 2008
(New York Times Business, August 8, 2008)
Chemogen assay results (FIND Tanzania study 2006)
Tessema, Svenson et al., 2001
On the way to a POC: The search for TB antigens
Clearview MTB ELISA: Result
overview for completed
studies (accepted or
submitted)
Site
Overall
sensitivity
Sensitivity in
HIV-infected
Sensitivity in HIV-
uninfected
Specificity
Mbeya 51% 65% 34% 93%
Dar Es Salaam 65% 65% 86%
Harare 44% 52% 21% 89%
Cape Town 1 59% 67% 14% 96%
Cape Town 2 38% 38% 100%
• Alternative LAM AB pairs in clinical validation studies in Zimbabwe/SA
• Several LAM prototype POC assays in development
• Promising new AG leads followed-up in several collaborations
Urinary LAM assay: Potential application in HIV-
positive patients?
What’s next:
01
23
4
LA
M o
ptical density a
bo
ve c
ontr
ol
10-50 50-200 200-350 >350
LAM ODs in CD4 groups for HIV+ and TB+ participants
01
23
4
LA
M o
ptical density a
bo
ve c
ontr
ol
10-50 50-200 200-350 >350
LAM ODs in CD4 groups for HIV+ and TB+ participants
01
23
4
LA
M o
ptical density a
bo
ve c
ontr
ol
10-50 50-200 200-350 >350
LAM ODs in CD4 groups for HIV+ and TB+ participants
01
23
4
LA
M o
ptical density a
bo
ve c
ontr
ol
10-50 50-200 200-350 >350
LAM ODs in CD4 groups for HIV+ and TB+ participants Box & Whisker plot of LAM
concentrations in TB patients
with and without HIV
coinfection.
Towards TB Serology:
Anti-M.tb Antibody Profiling
• Antigen array chip with ~4,000 proteins
• Whole M.tb proteome screen to identify a set of diagnostic antigens for seroprofiling
• >1,000 sera selected for WPS (FIND sample repository)
Countries of origin of study subjects
M. tuberculosis Whole Proteome
Screen: profiling 927 serum samples from 10
countries (FIND sample repository)
Data from different sites have been reconciled and re-coded in a
common semantic framework to create an Oracle database that
integrates microarray measurements and clinical data
Establishment of database
Original Sources Standardized
Oracle Database
w/ custom query interface
•All confirmed TB vs. endemic non-TB controls included in analysis
• Two independent statistical analyses employed
• 19 top rank proteins targeted for purification
•Validation studies to be initiated
Ranking of Proteins from Whole Proteome Screen
Giant pouched rats have been trained to detect TB in sputum specimens by smell
alone. Preliminary results of a blinded study using 2 rats for 67 AFB+ and 752 AFB-
samples found a sensitivity of 86% and a specificity of 89%
Background - Natural Olfaction
Breath
detection
Scensive Technologies
Application of E-Nose to Detection of M.
tuberculosis in Culture*
*Fend R, et al. JCM 2006;44:2039-45.
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